RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.

Aberrant methylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers. There are major smoke exposure, histology, geography and gender-related changes in non-small cell lung cancer (NSCLC). We investigated smoking-related, histologic, geographic and gender differences in the methylation profiles of resected NSCLCs. We examined 514 cases of NSCLC and 84 corresponding nonmalignant lung tissues from 4 countries (USA, Australia, Japan and Taiwan) for the methylation status of 7 genes known to be frequently methylated in lung cancers [p16, RASSF1A (RAS association domain family 1), APC, RAR␤, CDH13, MGMT and GSTP1]. Multivariate analyses were used for data analysis. Adenocarcinoma was the major histologic type in women and never smokers; analyses that involved smoke exposure and gender were limited to this histology. Our major findings are a) methylation status of any single gene was largely independent of methylation status of other genes; b) the rates of methylation of p16 and APC and the mean Methylation Index (MI), a reflection of the overall methylation status, were significantly higher in ever smokers than in never smokers; c) the mean MI of tumors arising in former smokers was significantly lower than the mean of current smokers; d) the methylation rates of APC, CDH13 and RAR␤ were significantly higher in adenocarcinomas than in squamous cell carcinomas; e) methylation rates of MGMT and GSTP1 were significantly higher in the USA and Australian cases than in those from Japan and Taiwan; and (f) no significant gender-related differences in methylation patterns were noted. Our findings demonstrate important smoke exposure, histologic type and geography-related differences in the methylation profiles of NSCLC tumors.

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BackgroundDNA methylation regulates together with other epigenetic mechanisms the transcriptional activity of genes and is involved in the pathogenesis of malignant diseases including lung cancer. In non-small cell lung cancer (NSCLC) various tumor suppressor genes are already known to be tumor-specifically methylated. However, from the vast majority of a large number of genes which were identified to be tumor-specifically methylated, tumor-specific methylation was unknown so far. Thus, the major aim of this study was to investigate in detail the mechanism(s) responsible for transcriptional regulation of the genes SPAG6 and L1TD1 in NSCLCs.MethodsWe analysed publically available RNA-sequencing data and performed gene expression analyses by RT-PCR. DNA methylation analyses were done by methylation-sensitive high-resolution melt analyses and bisulfite genomic sequencing. We additionally investigated protein expression using immunohistochemistry. Cell culture experiments included tumor cell growth, proliferation, viability as well as colony formation assays. Moreover, we performed xenograft experiments using immunodeficient mice.ResultsWe observed frequent downregulation of SPAG6 and L1TD1 mRNA expression in primary tumor (TU) samples compared to corresponding non-malignant lung tissue (NL) samples of NSCLC patients. We furthermore observed re-expression of both genes after treatment with epigenetically active drugs in most NSCLC cell lines with downregulated SPAG6 and L1TD1 mRNA expression. Frequent tumor-specific DNA methylation of SPAG6 and L1TD1 was detected when we analysed TU and corresponding NL samples of NSCLC patients. ROC curve analyses demonstrated that methylation of both genes is able to distinguish between TU and NL samples of these patients. Immunohistochemistry revealed a close association between SPAG6/L1TD1 methylation and downregulated protein expression of these genes. Moreover, by performing functional assays we observed reduced cell growth, proliferation and viability of pCMV6-L1TD1 transfected NSCLC cells. In addition, reduced volumes of tumors derived from pCMV6-L1TD1 compared to pCMV6-ENTRY transfected NCI-H1975 cells were seen in a xenograft tumor model.ConclusionsOverall, our results demonstrate that SPAG6 and L1TD1 are tumor-specifically methylated in NSCLCs and that DNA methylation is involved in the transcriptional regulation of these genes. Moreover, in vitro as well as in vivo experiments revealed tumor-cell growth suppressing properties of L1TD1 in NSCLC cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0568-5) contains supplementary material, which is available to authorized users.

SummaryIn contrast to its close homolog CDK4, the cell cycle kinase CDK6 is expressed at high levels in lymphoid malignancies. In a model for p185BCR-ABL+ B-acute lymphoid leukemia, we show that CDK6 is part of a transcription complex that induces the expression of the tumor suppressor p16INK4a and the pro-angiogenic factor VEGF-A. This function is independent of CDK6’s kinase activity. High CDK6 expression thus suppresses proliferation by upregulating p16INK4a, providing an internal safeguard. However, in the absence of p16INK4a, CDK6 can exert its full tumor-promoting function by enhancing proliferation and stimulating angiogenesis. The finding that CDK6 connects cell-cycle progression to angiogenesis confirms CDK6’s central role in hematopoietic malignancies and could underlie the selection pressure to upregulate CDK6 and silence p16INK4a.